CN115697088A

CN115697088A - Oil dispersion preparation rich in omega fatty acid

Info

Publication number: CN115697088A
Application number: CN202180039957.5A
Authority: CN
Inventors: 桑迪普·奥罗拉; 内哈·奥罗拉
Original assignee: Bioplus Life Sciences Pvt Ltd
Current assignee: Bioplus Life Sciences Pvt Ltd
Priority date: 2020-05-02
Filing date: 2021-05-03
Publication date: 2023-02-03
Also published as: EP4142521A4; US20230165794A1; AU2021267426A1; BR112022022230A2; EP4142521A1; WO2021224940A1

Abstract

The present invention comprises an oral omega-3 fatty acid rich oil emulsion composition comprising DHA 50-100mg/ml, made of all natural and biocompatible ingredients, for the management of the treatment of a disorder to overcome it. The composition may be a thixotropic emulsion in the nanometer size range with better absorption, better stability at room and refrigeration temperatures (2-8 ℃), alone or in combination with therapeutically effective amounts of: vitamins, minerals, natural ingredients Generally Regarded As Safe (GRAS). The disorders include premature labor disorders in pregnant women, cognitive disorders in children, and cardiovascular disorders. The emulsion is prepared by adding an oil phase comprising a natural emulsifier to an aqueous phase comprising: gums, vitamin E TPGS, preservatives, and high intensity sweeteners. The invention also encompasses High Performance Liquid Chromatography (HPLC) methods for the determination of omega-3 fatty acids.

Description

Oil dispersion preparation rich in omega fatty acid

Technical Field

The present invention relates to a stable and effective omega 3 fatty acid (O3 FA) -rich oil dispersion for oral administration in infants, children, adults and food fortification.

Background

Docosahexaenoic acid (DHA) is an essential omega fatty acid, found in neurons and other body tissues. Normal development of neural tissue and cognitive skills is highly dependent on adequate intake of omega-3 fatty acids, especially DHA, in the diet. As brain mass increases approximately 3.5-fold before the age of 5 years, it requires accumulation of omega-3 fatty acids (O3 FA). Thus, the American Dietary Association (American Dietary Association) strongly recommends the ingestion of O3FA (500 mg/day) from different Dietary sources during pregnancy and infancy and even in adulthood (Kris-Etherton, PM.and Innis, S.position of the American Dietary Association and Dietitian of Canada: dietary fasts, J.Am.Diet Association, 2007,107 (9), 1599-611). A large number of children worldwide suffer from cognitive disorders and undesirable neurological conditions, with Attention Deficit Hyperactivity Disorder (ADHD) being one of the most common cognitive disorders in childhood. In the United states, approximately 8-10% of children suffer from this disorder, and in India, the prevalence is even higher by 5-29% (http:// www. Slipsharhare. Net/adhdarabia/adhd-features-and-regulations). Furthermore, the increasing tendency to learning disability is an alarming problem for cognitive health in children; in the united states, about 240 million children suffer from this disorder (National center of learning disability, 2014). Omega fatty acids (O3 FA) are highly lipophilic in nature. For the purposes of this specification, "omega fatty acids" or "O3FA" are defined as fatty acids having at least one double bond in their carbon backbone. O3 FAs include, but are not limited to, omega-3 fatty acids and omega-6 fatty acids and omega-9 fatty acids or combinations thereof. Examples of O3FA include docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and/or arachidonic acid; or a combination thereof. Converting O3FA into water-soluble products to improve their applicability is a challenging task. Currently, available formulations such as soft gelatin capsules are the most commonly used formulations of O3FA rich oils, which have their own disadvantages; such as unpleasant reflux, flatulence, low bioavailability and inadequacy to infants and children. Furthermore, oral liquid formulations of O3FA rich oils are limited, and even these formulations further have poor dispersibility and absorption in GIT fluids, short shelf life, oxidative instability and poor sensory profile. Therefore, poor patient compliance and the lack of stable and therapeutically effective formulations in children remain major hurdles to the widespread clinical use of O3 FA-rich oils.

In the present invention, pharmaceutically stable and organoleptically elegant O3FA enriched oil emulsion formulations have been developed with increased bioavailability, oxidative stability, extended shelf life and improved therapeutic index. In addition, cell line studies demonstrate a safety profile.

Oral thixotropic emulsions in the nanometer size range of O3 FA-rich oils are described. The thixotropic emulsion composition of O3 FA-enriched microbial oil is stable and therapeutically effective. The invention also describes a process for the preparation of thixotropic emulsion compositions of O3 FA-enriched microbial oil. The composition is optionally fortified with vitamins and minerals.

O3FA is a biomolecule commonly and particularly involved in normal development of the brain and other nervous system disorders. O3FA deficiency is also associated with several medical conditions, including increased risk of preterm birth. However, due to its highly lipophilic, oily nature, O3FA has poor water dispersibility, poor sensory profile and oxidative instability. Currently, within the nutritional category, only limited oral formulations of O3FA rich oils are available. They have short shelf lives and poor oxidative stability along with limitations of oil separation during storage. In the present invention, stable thixotropic emulsion formulations for oral administration of O3FA were developed. The stable emulsion formulation is optionally fortified with vitamins and minerals. The therapeutic effectiveness and toxicity profile of the thixotropic emulsion formulation of the present invention has been demonstrated both in vivo and ex vivo.

Emulsions are biphasic colloidal systems comprising an oil phase, an aqueous phase, an emulsifier, and a stabilizer. O/W emulsion (oil-in-water) emulsions are the most suitable formulations for oral administration of lipophilic active moieties (i.e. drugs, oils, vitamins etc.) wherein the oil phase is well dispersed as oil spheroids in a continuous aqueous phase, stabilised by different emulsifiers and stabilisers. It was surprisingly found that although an emulsion of an O3FA enriched oil is physically unstable, wherein the oil separates after a period of time under storage, and it is also oxidatively unstable, upon conversion of the emulsion in a thixotropic system (gel-sol-gel), the resulting composition results in physical stability as well as oxidative stability, resulting in increased shelf life of storage. In addition, the delivery system has the flexibility to include different sweeteners and flavoring agents, which increases palatability during oral administration. Furthermore, this is easily upsized, since the preparation described therein is simple and easy. Thus, the present invention provides methods and systems in which palatable and stable compositions can be made from O3FA rich oils for use in nutritional therapy to obtain the health benefits of O3 FA.

DHA is one of the most widely used O3 FAs and plays an important role in the effective management of preterm birth disorders. In the present invention, the use of the developed dispersion formulation for the treatment of premature birth disorders has been disclosed.

Preterm birth (< 37 weeks gestation) is one of the leading causes of infant death worldwide. It accounts for about 17% of deaths in children under 5 years of age, and accounts for over 85% of all prenatal complications (Makrides M, best K. Docosa hexaenoic acid and preterm birth. Annals of Nutrition and metabolism.2016;69 (suppl. 1): 29-34.). Advances in antenatal and neonatal care will reduce the number of premature birth cases and improve cognitive impairment in infants. Epidemiological and randomized trial studies have observed long term pregnancy, infant weight at birth, and increased head circumference in populations with high fish consumption (Greenberg JA, bell SJ, van Ausdal w. Omega-3 fat acid recruitment shock. Reviews in observations in reviews in optics and gyencology.2008 (4): 162.;3. Bag ml, puralala se, messier se, prtchett dk, harris ws. White is the relationship between genetic tissue and cartilage acid (DHA) clinical acid and (ARA) vitamins. Protglandins, leukiness and Essential Fatty acids, sep 1-11). Olson and Joensen first observed that faradaic people consumed more long chain polyunsaturated fatty acids (LCPUFAs), such as seafood rich in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), with longer pregnancy durations and higher infant birth weights than danish people.

Despite the wealth of information on the pathophysiology of pregnant women and premature labour, there is currently no strategy available as a major prevention for a wide range of clinical applications. The prenatal period is a highly vulnerable window to DHA deficiency. Omega-3 fatty acids are essential and can only be obtained from the diet. These LCPUFAs including DHA are increasingly transferred from mother to fetus in the late gestation (3 rd gestation). Infants born prior to this transfer are at risk of defects. The need during pregnancy has not been established but may exceed the need for non-pregnant conditions. These fatty acids are important for fetal neural development, especially vision and neural function (Rogers LK, valentine CJ, keim SA. DHA filing: current innovations in pregnancy and childhood. Pharmacological research.2013Apr 1 (1): 13-9.. According to the National Institute of Health, the consumption of DHA is very low in both developed and underdeveloped countries. The average intake of children and adolescents is 40mg, and adults are about 90mg. Currently, the recommended set for DHA for pregnancy and lactation is at least 200-300 mg/day. However, some studies have shown that supplementation of less than 600 mg/day is not beneficial in preventing early preterm birth. DHA in the supplemental range of 600mg-1000mg is associated with a reduced risk of preterm birth and higher birth weight, as well as a positive impact on the brain development of the infant (https:// clinicaltrilials. Gov/ct2/show/NCT02626299term = DHA +800+ mg &/= predrm + birt &/& draw =2 &/& rand = 1).

Most pregnant women do not get enough omega-3 fatty acids because the primary dietary source of seafood is limited to two servings per week. In order for pregnant women to obtain sufficient omega-3 fatty acids, a variety of sources should be consumed, namely vegetable oil, weekly servings of low mercury fish, and supplements (fish oil or algae-based docosahexaenoic acid). In view of this new data, pregnant women of all dietary patterns may benefit from consuming between 600-1000 mg/day of DHA supplement from fish oil or algae oil. Algal oil has an advantage over fish oil in that it is derived from microalgae, which are substances consumed by fish to obtain their DHA and are biologically comparable to DHA available in fish oil.

Cognitive disorders are a type of mental health disorder that adversely affects the learning, memory, perception and problem solving abilities of children. These conditions range from profound impairment of intelligence to mild impairment of specific activities. These disorders result from low or inappropriate uptake of O3FA during the brain developmental stages of life. The most commonly used dosage form as a source of O3FA is a soft gelatin capsule enriched with O3 FA-rich oil. But they are not suitable for administration to infants and children and even to adults due to unpleasant reflux and low bioavailability. The additional limited availability and poor shelf life of liquid formulations with low levels of O3FA has prompted the need to develop stable and therapeutically effective formulations comprising an O3FA moiety.

Prior Art

U.S. patent 2006/0165735A1 A1 discloses an oil emulsion comprising: an oil component comprising polyunsaturated fatty acids; an emulsifier; an emulsion stabilizer; and water; wherein the oil emulsion is not heat treated. Physical stability at room temperature and 4C is claimed to be only 180 days. No additional storage requirements are made.

Us patent 2012/02516851 A1 discloses an oil-in-water emulsion comprising: a) An oil comprising polyunsaturated fatty acids; b) An emulsifier; c) Water; d) A metal chelator; and e) an antioxidant; wherein the metal chelator is present in an amount of about 3% to about 20% by weight of the emulsion, and wherein the antioxidant is present in an amount of about 2% to about 20% by weight of the emulsion. It is claimed that shelf life is up to five to six months under refrigerated conditions. High concentrations of metal chelating agents and antioxidants are used up to 20%, well above the specified limits.

Us patent 2011/0054029A1 A1 discloses a water-soluble dietary fatty acid gel formulation comprising: 1 to 75wt% of dietary fatty acids; and 25 to 99wt% of a nonionic surfactant. Hydrogenated castor oil/polyethylene glycol hydroxystearate (Cremophor RH 40) using a non-ionic surfactant may have health related side effects such as vasodilation, nephrotoxicity, etc. when consumed at high concentrations.

US 20120093998A1 discloses an emulsion comprising (i) 5-20 wt.% (wt-%), based on the total weight of the emulsion, PUFA, and (ii) 10-40wt-%, based on the total weight of the emulsion, of at least one emulsifier, which emulsifier is a polymeric hydrocolloid derived from plant sources, (iii) 5-45wt-%, based on the total weight of the emulsion, of at least one adjuvant, and (iv) 15-50wt-%, based on the total weight of the emulsion, of water. However, no details are given of spheroid size, physical and chemical stability of the prepared formulation.

US 9302017B2 discloses micellar formulations of omega 3 fatty acid esters having an average diameter of about 1 to 10 μm. The micelle formulation was based entirely on polysorbate 80 and Pluronic F87, synthetic surfactants using fish oil. The finished preparation has no detailed physical and chemical stability and also no disclosure of the strength in terms of omega 3 fatty acids. The inventors claim the use of the formulation in the maintenance of cardiovascular health.

US 2011/0200644A1 discloses an emulsion comprising an emulsifier, an isotonizing agent and an oil comprising ethyl docosahexaenoic acid (DHA-EE), wherein the emulsion is substantially free of eicosapentaenoic acid (EPA) and suitable for parenteral administration and the O3FA ester emulsion formation is based on gelatin and the lipid E80 SN. The prepared formulation was not subjected to detailed physical and chemical stability. The inventors claim the use of the formulation in the maintenance of inflammatory conditions.

Us patent 2012/0308704A1 discloses an emulsion as an ingredient or additive for producing a food product comprising omega-3 fatty acids, the emulsion comprising: an external aqueous phase comprising at least one water-soluble antioxidant dissolved in water; and an inner fat or oil phase comprising vegetable oil globules provided with at least one fat or oil soluble antioxidant and provided with an omega-3 fatty acid ester, wherein the vegetable oil globules are provided with a shell made of vegetable protein. The emulsion is based on the use of pea protein isolate as emulsifier and fish oil as source of omega 3 fatty acids. The formulations developed were primarily claimed for food fortification, in particular for the production of poultry beef sausages with a content of 1% omega 3 fatty acids.

Us patent 9532963B2 discloses a food supplement or nutritional supplement composition comprising: a fatty acid oil mixture comprising from about 25% to about 75% eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by weight of the fatty acid oil mixture, wherein the EPA and DHA are in a form selected from the group consisting of methyl esters, ethyl esters, and triglycerides; and at least one free fatty acid selected from: EPA, DHA, ALA, HPA, DPA, ETA, ETE, STA, linoleic acid, GLA, AA, osbond acid, oleic acid, ricinoleic acid, sinapic acid and mixtures thereof. The formulations are entirely based on synthetic surfactants such as Cremophor, pluronic, briz. The inventors claim the use of the formulation in the maintenance of cardiovascular health.

Thus, in none of the prior art, the problem of preterm birth has been investigated or solved.

None of the emulsions carries DHA concentrations above 25mg/ml, which is required for therapeutic efficacy in children within a few ml. Unless a therapeutically effective dose can be carried in a reasonably small amount of emulsion, it is not useful as a practical dosage form. Furthermore, the prior art compositions have synthetic surfactants, which prohibit administration in children, including newborns. Furthermore, the dispersed phase of the prior art emulsions is in the micron range, which has a limited absorption of DHA. The prior art emulsions have poor stability at room temperature. Furthermore, the preparation process is very cumbersome and expensive.

Therefore, there is a need for improved emulsions with better and improved manufacturing processes.

Disclosure of Invention

The invention includes oral administration of an omega-3 fatty acid rich oil emulsion composition for the management of treatment of disorders to overcome said disorders. The omega-3 fatty acid rich oil may be a microalgae oil comprising 40% DHA and the emulsion comprises DHA in an amount of 50-100mg/ml.

It is an embodiment of the present invention that said oral omega-3 fatty acid rich oil emulsion is made of ingredients all of which are natural and biocompatible and is suitable for administration to children including newborns.

In one embodiment, the oral omega-3 fatty acid rich oil emulsion composition has a dispersed phase in the nanometer size range.

Also claimed is the invention, wherein the oral omega-3 fatty acid rich oil emulsion composition has stability at room temperature (about 30 ℃) and refrigeration temperature (2-8 ℃).

In another embodiment, an oral omega-3 fatty acid rich oil emulsion composition comprises therapeutically effective amounts of vitamins and minerals.

Disorders treated by oral administration of omega-3 fatty acid rich oil emulsions include but are not limited to preterm birth disorders in pregnant women, cognitive disorders in children and cardiovascular disorders.

Oral omega-3 fatty acid rich oil emulsions of the invention, preterm labor impairment in pregnant women is overcome by achieving normal labour, cognitive impairment in children is overcome by improving their cognitive abilities, and cardiovascular disorders are overcome by restoring health.

In another embodiment, the present invention comprises an oral omega-3 fatty acid rich thixotropic emulsion of oil in the nanometer size range with better absorption. Oral administration of omega-3 fatty acid rich oils can be a thixotropic emulsion in the nanometer size range with higher surface area and absorption. The oral omega-3 fatty acid rich thixotropic emulsion of oil comprises a natural emulsifier and derivatives thereof and a biosurfactant, alone or in combination: vitamins, minerals, natural ingredients Generally Regarded As Safe (GRAS). Natural emulsifiers include, without limitation, one or more of natural gums, clays, polymers, and the like; the additives include one or more selected from the group consisting of: rheology modifiers, antioxidants, preservatives, stabilizers, sweeteners and flavouring agents.

The invention also includes an oral omega-3 fatty acid rich oil emulsion composition having a dispersed phase with nanometer dimensions comprising natural emulsifiers and their derivative surfactants, alone or in combination: vitamins, minerals, natural ingredients Generally Regarded As Safe (GRAS).

The invention also encompasses High Performance Liquid Chromatography (HPLC) methods for the determination of omega-3 fatty acids. The method comprises the following steps: the blank, standard solution-1, standard solution-2 and sample solution were injected separately into the chromatograph, the chromatogram recorded and the peak response of docosahexaenoic acid (DHA) measured. The blank injected was a single repeat, the standard solution injected-1 was five repeats, the standard solution injected-2 was two repeats, and the sample solution injected was a single repeat, the column used was Thermo Syncronis C18 (250x 4.6 mm) -5 μm or equivalent, the pump mode was isocratic, the flow rate was 1.0ml/min, detection was at UV 210nm, the injection volume was 20 μ l, the column oven temperature was 45 ℃, and the run time was 20 minutes. The solution-1 comprises a DHA working standard, and the solution-2 comprises a DHA test solution. The sample solution comprised a known amount of algae oil rich in DHA sonicated with n-heptane in a round bottom flask for a period of time, to which was added methanolic sodium hydroxide solution and refluxed with a stirrer for 10 minutes, cooled in an ice bath without removing the round bottom flask, slowly and carefully added boron trifluoride methanolic complex solution, further refluxed with a magnetic stirrer solution, cooled in an ice bath without removing the round bottom flask, carefully slowly added n-heptane and refluxed, cooled mixture and removed the round bottom flask, added saturated sodium chloride solution, shaken up and transferred the contents to a centrifuge tube, centrifuged at low speed, diluted with isopropanol to release the heptane layer and mix it, further diluted the solution with methanol and mix it.

The natural emulsifier comprises one or more selected from the group consisting of: (i) Vitamin ETPGS (d- α -tocopheryl polyethylene glycol 1000 succinate); (ii) Phospholipids including one or more selected from the group consisting of soybean phosphatidylcholine and egg yolk phosphatidylcholine, distearoyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine; gums including one or more selected from the group consisting of gum arabic, guar gum, xanthan gum, and gum tragacanth (targacanth); a polymer including one or more selected from the group consisting of pectin, gelatin, and algin; an emulsion stabilizer comprising one or more selected from the group consisting of xanthan gum, guar gum, gum arabic, bentonite, glycerin and mixtures thereof.

The oral omega-3 fatty acid rich oil comprises one or more selected from the group consisting of microalgae oil, fish oil or linseed oil. Antioxidants including one or more selected from the group consisting of butylated hydroxytoluene, rosemary oil, sodium ascorbate, vitablend (consisting of vitamin E and ascorbyl palmitate), sodium metabisulfite (sodium metabisulfite), ascorbyl palmitate, and vitamin E; vitamins selected from the group consisting of oil soluble vitamin A, vitamin D, vitamin E, and vitamin K; one or more of the group consisting of water-soluble vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, folic acid and vitamin C. Minerals including zinc, copper, magnesium, potassium, calcium such as calcium phosphate or carbonate, iron, and beta-carotene; a buffer comprising one or more selected from the group consisting of sodium carbonate citrate and phosphate buffer; a flavoring agent comprising one or more flavorings selected from the group consisting of orange, strawberry, raspberry, mango, peach, vanilla, lime flavorings; a sweetener comprising one or more selected from the group consisting of sorbitol, xylitol, mannitol, sucralose, stevia, aspartame, neotame (Neotame), acesulfame potassium, and mixtures thereof; preservatives, including rosemary extract, sodium benzoate, sodium azide, methyl paraben and propyl paraben.

The invention also discloses a process for preparing an oral omega-3 fatty acid rich oil emulsion, comprising the following steps: (a) Preparing an oil phase of the microalgae oil enriched in omega-3 fatty acids by mixing, at room temperature, a natural emulsifier, a vitamin blend comprising antioxidants and the microalgae oil enriched in DHA in a manufacturing tank with a stirrer, (b) preparing an aqueous phase in a tank with a stirrer, comprising the following steps: (ii) dissolving vitamin E TPGS (d- α -tocopheryl polyethylene glycol 1000 succinate) in another vessel with mechanical agitation and mixing other water-soluble ingredients including preservatives and high intensity sweeteners with this solution, (iii) thereafter mixing the gum and both the vitamin E and ascorbyl palmitate solutions with mechanical agitation for a period of time required to form a homogeneous mixture, (iv) thereafter adding the oil phase to the aqueous phase with mechanical agitation, maintaining the two phases at room temperature, (v) adding the flavoring agent, and continuing to stir for an additional period of time.

Process for the preparation of an oral omega-3 fatty acid rich oil emulsion according to claim 17 wherein the natural emulsifier comprises soy phosphatidylcholine and the oil phase comprises a mixture of: (I) Omega-3 fatty acid enriched microalgae oil 12.5-25% w/v, (II) antioxidant blend (vitaund) ^TM ) 0.05 to 0.5%, (III) butylated hydroxytoluene 0.1%, (IV) DHA-rich microalgae oil 12.5-25 w/V, (V) manufacturing tank is stainless steel jacketed, (VI) stirring at 40-50 ℃ with stirring speed of 100-300RPM, (VII) the pectin is 0.3-1.5% w/V) xanthan gum, (VIII) soaking in purified water at 40-50 ℃ for 1-5h period, (IX) enriching vitamin E TPGS (d- α -tocopherol polyethylene glycol 1000 succinate) at 1-5 w/V) with mechanical stirring at 1000-1500RPM, sodium benzoate 0.02-0.1 w/V, preservative X) high intensity sweetener is sucralose 0.1-0.5 w/V, (XI) keeping the two phases of the mixture under mechanical stirring at 40-50 ℃ at 1000-1500RPM and mixing the pectin and vitamin E, and the two phases of tpe 0.1-0.5 w/V, (XII) keeping the oil phase mixture under stirring uniformly at 40-50 ℃ and keeping the oil phase under stirring at 1000-1500RPM, and stirring at 30 min and finally keeping the two phases of the oil phase under stirring at 1500 w/V.

Detailed Description

The inventors of the present invention found that the need to develop stable and therapeutically effective formulations comprising O3FA moieties can be met by incorporating O3FA rich oils into colloid/dispersion systems with nanometer dimensions for efficient absorption across the gastrointestinal tract. In one embodiment, the present invention comprises an emulsion (wherein the concentration of DHA is 50-100 mg/ml) with a conventional microalgae oil (containing 40% DHA).

In one embodiment of the invention, the emulsion comprises therapeutically effective amounts of vitamins and minerals.

Still further, the delivery system includes one or more stabilizers, one or more emulsifiers, one or more antioxidants, one or more sweeteners, one or more flavoring agents, and a carrier.

A further embodiment of the invention is the use of only natural surfactants. Synthetic surfactants are not used and therefore the emulsion composition is beneficial for administration to children, including newborns.

In a further embodiment of the invention, the dispersed phase of the emulsion is in the nanometer range. This makes the emulsion better available for absorbing DHA.

In a further embodiment, the emulsion of the invention has good stability at room temperature (about 30 ℃ and also at refrigeration temperatures (2-8 ℃).

In addition, the present method of preparing an emulsion is very simple and cost effective compared to the prior art methods of manufacturing DHA-enriched oil emulsions (including microalgae oils having 40% DHA).

The O3 FA-rich oil is preferably microalgae, but may also include other O3 FA-rich oil sources, such as fish oil and other sources of O3 FA.

Examples of emulsifying agents include vitamin E TPGS (d- α -tocopheryl polyethylene glycol 1000 succinate), phospholipids, but are not limited to soy and egg phosphatidylcholine, distearoyl phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, gum arabic, guar gum, xanthan gum, tragacanth gum, pectin, gelatin, algin, bentonite. For the purposes of the present invention, an emulsifier can be defined as any molecule having a Hydrophilic Lipophilic Balance (HLB) value between 4 and 18.

The emulsion stabilizer may act as a thickener which prevents coalescence of oil droplets when used alone and when used in combination with a rheology modifier. Emulsion stabilizers include, but are not limited to, xanthan gum, guar gum, gum arabic, bentonite, glycerin, and mixtures thereof.

Antioxidants for preventing the autoxidation of polyunsaturated fatty acids include rosemary oil, sodium ascorbate, vitabind, sodium metabisulfite, ascorbyl palmitate, vitamin E, and the like.

Vitamins include, but are not limited to, oil soluble vitamin a, vitamin D, vitamin E, vitamin K; water-soluble vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, folic acid and vitamin C. Minerals include zinc, copper, magnesium, potassium, calcium such as calcium phosphate or carbonate, iron, and beta-carotene.

Buffering agents include, but are not limited to, sodium citrate, sodium carbonate, for maintaining the pH of the emulsion formulation.

Sweetening agents and flavoring agents that enhance the palatability of the oral emulsions include, but are not limited to, orange, strawberry, raspberry, mango, peach, vanilla, lime flavor, and sorbitol, xylitol, mannitol, sucralose, stevia, aspartame, neotame, acesulfame potassium, and the like, as well as mixtures thereof, alone.

The stability of oral O3FA rich oil emulsions was assessed at different storage temperatures and time points (0, 30, 60, 90 and 180 days) by assessing parameters of DHA content, specific gravity, pH, peroxide value, rheology studies, peroxide and oxidative rancidity (rancimat) analysis, and the therapeutic effectiveness of oral O3FA rich oil emulsions was assessed by rheology studies, peroxide and oxidative rancidity analysis, in vivo cognitive animal models and ex vivo Caco2 and Fr2 cell line studies.

No information is available in the prior art about the enhanced shelf-life and therapeutic effectiveness of oral O3FA rich oil emulsions. In the present invention, the benefits of O3 FA-enriched oil emulsion formulations are enhanced by preparing oral compositions of microalgae oil emulsions with enhanced storage and oxidative stability. The emulsions of the present invention are developed especially for the treatment of preterm birth disorders in pregnant women and cognitive disorders in removed children. The emulsions of the present invention have been shown to be effective in enhancing O3FA plasma levels compared to prior art compositions.

The invention discloses a composition of O3 FA-rich oil emulsion, which is stable in physical properties and also has oxidation stability. The present invention also discloses a process for preparing a pharmaceutically acceptable and stable O3 FA-rich oil emulsion formulation comprising a solid self-nanoemulsifying drug delivery system, a conventional emulsion, a micellar dispersion alone and in combination with: vitamins and minerals are associated with GRAS (generally regarded as safe) certified ingredients. The composition of the O3 FA-rich oil emulsion comprises different natural emulsifiers, such as natural gums and derivatives thereof and biosurfactants, alone or in combination. The composition also contains optimized amounts of additives such as rheology modifiers, antioxidants, preservatives, stabilizers, sweeteners, flavoring agents, and the like. The effect of the type and amount of emulsifiers, stabilizers and antioxidants on oxidative stability, shelf life, rheology and toxicity profile was investigated by peroxide number, rheological analysis, cell line and animal studies, respectively. The resulting emulsion has enhanced shelf life and is resistant to oxidation and coalescence. The use of the developed formulations for the treatment of omega 3 fatty acid deficiencies in humans or animals and for reducing the risk of humans associated with preterm birth or cognitive disorders or for cardiovascular health is disclosed. In addition, a new simple, accurate and precise HPLC method for the determination of major omega 3 fatty acids is disclosed in the present invention.

The invention also encompasses novel High Performance Liquid Chromatography (HPLC) methods for analyzing DHA content in a sample. The current method of the Official Association of Agricultural Chemists (AOAC) is Gas Chromatography (GC) with a Flame Ionization Detector (FID). The relevant standard deviation for the GC analysis method was 5%, whereas for the HPLC method of the invention the relevant standard deviation was less than 2%. The GC method is less reproducible and the HPLC method of the present invention is very reproducible. The stability of the sample solution was good in the HPLC method of the invention, but poor in GC.

Illustrative examples of the compositions of the present invention are provided below and studied. The examples are illustrative only and do not limit the scope of the claims, which are obvious to a person skilled in the art and obvious equivalents to a person skilled in the art.

Drawings

FIG. 1: transmission Electron Micrographs (TEMs) of the emulsion formulations are illustrated.

FIG. 2: spheroid size analysis was performed on emulsion formulations using a dynamic light scattering method.

FIG. 3: the oil preparation (A) rich in O3FA is shown at 40s ^-1 At a constant shear rate of (B) in the range from 0 to 60s ^-1 Rheological analysis at variable shear rate (C) thixotropic analysis of the 03FA rich oil emulsion.

FIG. 4: the peroxide values of O3 FA-enriched oil emulsion A) under real-time conditions B) under refrigerated conditions are shown graphically.

FIG. 5 is a schematic view of: the induction time for the formation of secondary oxidation products in oxidative rancidity analysis in emulsions (induction time 2.57 h) is graphically shown compared to neat oil (induction time 0.45 h), indicating a higher oxidative stability of the emulsions.

FIG. 6: the biological safety of emulsion formulations in the MTT assay against A) Caco-2 cell line B) Fr2 cell line is illustrated.

FIG. 7: a) paracellular permeability B) TEER measurement of emulsion formulations is illustrated. (both of these parameters are a measure of the changes in the tight junctions and paracellular spacing of the gastrointestinal tract cells).

FIG. 8: the permeability of DHA in the DHA emulsion formulation compared to DHA oil.

FIG. 9: the pharmacokinetics of the DHA emulsion formulation were optimized by measuring the DHA content in the phospholipids, compared to DHA oil.

FIG. 10: HPLC chromatograms of the method for estimating DHA were developed and validated.

FIG. 11: process flow diagram for commercial production of DHA emulsion.

Example 1

Preparation of O3 FA-enriched oil emulsion preparation

An emulsion is prepared from microalgae oil rich in O3FA, wherein the microalgae oil accounts for 5-25% of the total content of the emulsion. In a first step, the oil phase is prepared by: mixing 0.4% w/v soybean phosphatidylcholine, 0.05% Vitablend, 0.1% butylated hydroxytoluene, and 12.5-25% w/v DHA-enriched microalgae oil in a stainless steel jacketed manufacturing tank with a stirrer at 40 deg.C and a stirring speed of 100 RPM. In the second step, the aqueous phase was prepared in a stainless steel jacket manufacturing tank with a stirrer. The xanthan gum (0.4% w/v) was soaked in purified water at 40 ℃ for a period of 2-3 h. In another vessel, the vitamin ETPGS (2% w/v) was dissolved and the other water-soluble ingredients sodium benzoate (0.05% w/v) and sucralose (0.1% w/v) were mixed with the solution, under mechanical agitation at 1000-1200RPM at 40 ℃. Both the gum and the vitamin E TPGS solution were then mixed for 30 minutes at 40 ℃ with mechanical agitation at 1000-1200RPM to form a homogeneous mixture. Finally, the oil phase was added to the water phase with mechanical stirring at 1000-1200rpm, maintaining both phases at 40 ℃. After adding orange oil (0.5% w/v) as a flavoring agent, stirring was continued for 0.5-1.0 hours. A process flow diagram for a commercial production concentration of DHA emulsion of 50-100mg/ml is shown in FIG. 9.

Table 1: composition of O3 FA-enriched oil emulsion formulation at a concentration of 50mg/ml (prototype 1)

Table 2: composition of O3 FA-enriched oil emulsion preparation at 75mg/ml (prototype 2)

Table 3: composition of O3 FA-enriched oil emulsion preparation at 100mg/ml (prototype 4)

Table 4: composition of O3 FA-enriched oil emulsion formulation with vitamins using natural emulsifiers in combination (prototype 4)

Example 2: preparation of O3 FA-enriched oil emulsion fortified with vitamins and minerals

A method of making an alkaline emulsion is provided as in example 1, to which vitamins and minerals and folic acid are added. The composition of the product is given in tables 5 and 6.

Table 5: composition of O3 FA-enriched oil emulsion preparation fortified with vitamins and minerals

Table 6: composition of O3 FA-enriched oil emulsion preparation fortified with vitamins and minerals

Example 3: in vitro characterization for quality control testing

The following are the studied/determined properties/characteristics and the results are provided in table 7 below.

Spheroid size and zeta potential analysis

The spheroid size, size distribution and zeta potential of the emulsions were measured on O3FA rich oil emulsions prepared in the examples by diluting the samples 100-fold with triple distilled water (triple distilled water) using a particle size analyzer (Malvern Zetasizer Nano ZS90, UK). In addition to the average particle size, the intensity distribution and polydispersity index (PDI) of the particles were measured, which is a measure of the uniformity of the size distribution.

pH of emulsion formulation

O3 FA-rich oil emulsions for oral administration have been developed; the pH of the emulsion should therefore be within the acceptable range required for oral administration, i.e. between

pH

5 and 8. The pH of the prepared emulsion formulation was measured using a pH meter (Toshcon CL-54).

Transmission Electron Microscope (TEM) analysis

The size and shape of the dispersed phase in the emulsion system was observed by operating a Transmission Electron Microscope (TEM) (HRTEM, JEM 2100, JEOL, japan) at an acceleration voltage of 200kV with a beam current of 100. Mu.A. The sample was diluted with triple distilled water at a ratio of 1. The grid was air dried and observed under a transmission electron microscope. The results are provided in figure 1.

Study of thermodynamic stability

The thermodynamic stability of O3 FA-enriched oil emulsion formulations was determined by storing emulsion samples at different temperatures of 4 + -1 deg.C and 45 + -1 deg.C for 24h at the respective temperatures. Finally, after the cooling and heating cycles, the samples were subjected to centrifugal stress at 3000rpm for 10min and monitored for the extent of any oil separation.

Viscosity of the solution

The viscosity of the prepared emulsion was determined by a Brookfield viscometer using a No. 5 spindle at a constant shear rate of 40 rpm/min.

Dispersion test and dilution robustness

Five ml of the prepared formulation was added to 500ml of distilled water in a USP type II dissolver (Lab india DS 8000) at 37 ± 0.5 ℃ and 50 rpm. Immediately after addition to the container, the water dispersibility of the formulation was visually inspected using the following grading system: grade a-fast dispersing milky emulsion (< 1 min); grade B-medium dispersed milky emulsion (> 2 min); and a grade C: the milky emulsion was slowly dispersed (> 5 min), oil appeared. Dilution robustness is an important parameter of emulsions to ensure that emulsions prepared after oral administration have similar properties at different dilutions. 1ml of each emulsion was diluted to 100 and 1000 fold with distilled water and 0.1N HCl. The diluted emulsion was observed for 24h to determine the separation of the oil phase at higher dilutions and spheroid size. Dilution robustness is an important parameter for understanding the behaviour of emulsion spheroids under in vivo conditions. The oil phase separates resulting in intestinal malabsorption. The results obtained are provided in table 7.

Table 7: in vitro characterization of O3 FA-enriched oil emulsion formulations

Example 4

Rheological analysis

Rheology is an important parameter for assessing the physical stability and thixotropic behavior of emulsions subjected to different shear rates and stresses. The viscosity and thixotropic profile of the O3FA emulsion were determined by using a rheometer (Rheolab QC, anton paar, germany). At a constant shear rate (40 s) at a temperature of 25 deg.C ^-1 ) And varying shear rates (0-60 s) ^-1 ) To determine the viscosity. At 80s ^-1 The thixotropic behaviour of the O3FA emulsion was determined at shear rates to evaluate the strength recovery ratio of the emulsion prepared after applying and removing the shear stress. The results obtained are given in table 8 and fig. 3.

Table 8: rheological behavior of emulsion formulations.

Example 5

Peroxide value estimation

Peroxides are the main oxidation products formed during the oxidation of oils and lipids. They were measured at time intervals of 0, 30, 60 and 90 days according to the official method followed by the Association of Analytical Community (AOAC). During the present study, samples were stored under accelerated storage conditions and refrigerated storage conditions. An acetic acid-chloroform mixture (30 mL) in a ratio of 3. 0.5mL of a saturated solution of Potassium Iodide (PI) was added, and the Erlenmeyer flask was placed in the dark and shaken for 1min at random times. 0.5mL of 1% w/v freshly prepared starch solution was added and titrated with 0.01N sodium thiosulfate, shaking vigorously until the blue color disappeared. The peroxide value is calculated by using the following equation. The results obtained are given in figure 4.

S = titration volume of sample

B = blank titration volume

Example 6

Analysis of oxidative rancidity

At ambient temperature, taste and odor deterioration (rancidity) due to oxidation is a slow process; oxidative rancidity methods accelerated the natural autoxidation process and measured the oxidative stability of the product over time. Measured at a temperature of 90 ℃ using an 892Professional Rancimat apparatus from Metrohm, heiheis at an air flow rate of 20mL of air/h. 3ml of the emulsion formulation was kept in a reaction vessel attached to a measuring vessel and an air flow tube. The formed secondary metabolite is transferred with the gas flow into the measuring vessel and the change in the conductivity of the measuring solution is measured as an inflection point. The results are provided in fig. 4.

Example 7

Stability study

Accelerated stability tests were performed according to ICH (International Conference for harmony) guidelines by: the O3FA enriched emulsion was stored in sealed amber bottles under refrigerated conditions (2 to 8 ℃) and under accelerated conditions (40 ℃/75% RH), real time (25 ℃/75% RH) by using a stable chamber and the results are provided in table 9.

Table 9: results of stability studies in real time and under refrigerated storage conditions.

All values are expressed as mean ± SD (n = 3). "-" indicates a significant change in the parameter, where p <0.05.

Example 8

Cell cytotoxicity assay

Cell viability assays were performed in human colorectal (Caco 2) and normal mammary epithelial (Fr 2) cell lines using the human colorectal cell line (Caco 2) with standard MTT (3- (4, 5-dimethylthiazol-2-yl) -2, s-diphenyltetrazolium bromide) method. Both cell lines were seeded in 96-well plates and Caco2 cell lines were treated with O3FA concentrations of 10, 20, 40, 80, 100 and 200 μ M in emulsion formulations for 24 hours, while Fr2 was treated with O3FA concentrations of 25, 12.5, 6.25, 3.12, 1.56 and 0.78mg/ml in emulsion formulations for 24 hours. After treatment of the O3 FA-enriched oil, the medium of each well was supplemented with 20. Mu.L of MTT [3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide ] reagent and allowed to incubate for an additional 4h. The formazan crystals formed were dissolved by adding 200 μ L of DMSO (dimethyl sulfoxide) to each well. Purple crystals were visible in the proportion of viable cells and absorbance was obtained at 570nm in a microplate reader (model: omega fluorostar, BMG Labtech Ltd., germany). The results of% cell viability are summarized in fig. 6 and it is clearly shown that the DHA emulsion formulations developed have a cell viability higher than 90% at all treatment concentrations

Example 9

Resistance across membrane

At 37 ℃ with 5% CO ₂ Supply toIn a wet incubator, a human colorectal cell line (Caco-2) was cultured in Dartbox Modified Eagle's Medium (DMEM) high sugar supplemented with 10% FBS and 10% antibiotic antifungal agent. When cells were grown to 60-70% confluence, and cells were harvested with trypsin-EDTA (0.25%), and at 2X 10 ⁵ cells/mL were seeded at a density of 0.4 μm pore size on polycarbonate membrane chamber (Transwell) inserts. Cells were cultured for 14 days to achieve differentiation and growth medium was refreshed every 2-3 days. Differentiated Caco-2 cells were treated with two concentrations of the optimized formulation (200. Mu.g/mL) and lipopolysaccharide (1. Mu.g/mL) as positive controls. After a 48h incubation period, TEER was measured for the control group using an epithelial volt-ohm meter with chopsticks electrodes (Millicell ERS-2, emd millipore, billerica, ma). By dividing the resistance value by the active membrane area (4.52 cm) ² ) The resistance per unit area is calculated. The observation results are shown in fig. 7. The TEER values treated with the developed emulsion formulation and microalgae DHA oil did not show any significant change compared to the negative control group, whereas significant TEER values (p) were observed for the positive control group (lipopolysaccharide)<0.001 Decrease (FIG. 7). Thus, it was confirmed that the developed formulations did not alter the integrity of the cell barrier of Caco-2 cells

Example 10

Determination of paracellular permeability

After measuring the TEER value, treated Caco-2 cells were additionally analyzed for paracellular permeability by using fluorescein isothiocyanate dextran (FD, 4 kDa). FD was dissolved in Phosphate Buffered Saline (PBS) to a concentration of 1mg/mL. 0.2mL of dye was added to the apical compartment of each insert, while 1.0mL of PBS was added to the basolateral well. The plates were covered with foil to prevent light inactivation of the fluorescent markers and placed in a shaker incubator at 150rpm at 37 ℃. After 12h, 0.3mL aliquots were removed from the basolateral chamber and fluorescence intensity was measured using a black 96-well plate in a multi-plate reader (Fluostar Omega, BMG Lab tech, germany). The observation results are shown in FIG. 7

Intestinal permeability measurement

The study was performed in the goat ileum for the developed emulsion formulation and DHA oil. The tissue was separated and cleaned with taiwanese pH 7.4. The thread was tied to one end of the ileum and placed in a student organ bath (student organ bath) containing tai chi fluid at pH 7.4. To prevent peristaltic muscle contraction, the liquid solution was bagged and a weight of 1g was attached to the unlined portion of the rod. The aeration and bath temperature around the ileal pouch was set at 37 ± 0.5 ℃. Samples were taken from the organ tubes at different intervals of 0.25, 0.5, 1, 2, 4 and 5h and the DHA content was measured. The results are shown in fig. 8.

Pharmacokinetic Studies

Pharmacokinetic studies over a 24h period were performed with the DHA emulsion formulation compared to DHA oil and a 2.2-fold increase in DHA uptake from the developed DHA formulation was observed (fig. 9). The increase of DHA in plasma was described by analysis of DHA content by gas chromatography. The improvement in DHA plasma levels observed during the pharmacokinetic studies was due to the increased permeability of DHA from the gut, which is clearly demonstrated from the results of ex vivo gut permeability studies.

Example 11: development and validation of HPLC assays for omega 3 fatty acids

A new HPLC assay method was developed with the following chromatographic conditions optimized as specified in table 10.

Table 10: HPLC advantage over GC analysis of DHA-enriched oil emulsions and DHA content in oils

Chromatographic conditions are as follows:

column: thermo Syncronis C18 (250x 4.6 mm), 5 μm or equivalent

Pump mode: constant gradient

Flow rate: 1.0ml/min

And (3) detection: UV,210nm

Injection volume: 20 μ l

Column furnace temperature: 45 deg.C

Operating time: 20 minutes

The optimized method is found to accurately measure the DHA content in the experimental scheme, the accuracy is high, the following parameters are verified, and the RSD value is less than 2%.1: specificity and recognition, 1.1: forced degradation research; 2: solution stability, 3: linearity, 4: accuracy, 5: accuracy-5.1: system accuracy, 5.2: method accuracy/repeatability, 5.3: intermediate precision/coarse, 6: range, 7: robustness-7.1: effect of flow rate variation, 7.2: detecting the effect of wavelength change, 7.3: influence of change in column furnace temperature, 8: and (4) system applicability.

Preparation of standard solution:

accurately weigh 250mg of DHA working standard (625 mg microalgae oil enriched in 40% DHA) into a round-bottomed flask, add 10mL of n-heptane by using a pipette, and sonicate for 15 minutes with intermittent shaking. 20mL of 0.5N methanolic sodium hydroxide solution was added and connected to the condenser via a Claisen adapter. The contents were refluxed with a magnetic stirrer for 10 minutes and cooled in an ice bath for 5 minutes without removing the round bottom flask. 20mL of boron trifluoride methanol complex solution (13-15%) are slowly and carefully added through a Claisen adapter and refluxed with a magnetic stirrer for another 30 minutes. Cooled in an ice bath for 5 minutes without removing the round bottom flask. 10ml of n-heptane were slowly and carefully added through a Claisen adapter and refluxed for a further 5 minutes. The mixture was cooled and the round bottom flask was removed. 5mL of saturated sodium chloride solution was added, shaken up and the contents transferred to a centrifuge tube. Centrifuge at low speed (500 RPM) for 5 minutes. The 2mL heptane-free layer was diluted to 50mL with isopropanol and mixed. Separately, 5mL of this solution was diluted to 50mL with methanol and mixed.

Preparation of sample solution: (DHA-enriched oil emulsions)

A sample corresponding to 250mg of DHA-rich oil emulsion was weighed accurately, placed in a round bottom flask, 10mL of n-heptane was added by using a pipette and sonicated for 15 minutes under intermittent shaking. In the illustrative work, microalgae oil rich in DHA was used; however, any other DHA-rich oil may be used. 20mL of 0.5N methanolic sodium hydroxide solution was added and connected to the condenser via a Claisen adapter. The contents were refluxed with a magnetic stirrer for 10 minutes and cooled in an ice bath for 5 minutes without removing the round bottom flask. 20mL of boron trifluoride methanol complex solution (13-15%) are slowly added cautiously through a Claisen adapter and refluxed with a magnetic stirrer for another 30 minutes. Cooled in an ice bath for 5 minutes without removing the round bottom flask. 10ml of n-heptane were slowly carefully added via a Claisen adapter and refluxed for a further 5 minutes. The mixture was cooled and the round bottom flask was taken out. 5mL of saturated sodium chloride solution was added, shaken up and the contents transferred to a centrifuge tube. Centrifuge at low speed (500 RPM) for 5 minutes. The 2mL upper heptane layer was diluted to 50mL with isopropanol and mixed. Separately, 5mL of this solution was diluted to 50mL with methanol and mixed.

Sample solution: (DHA-enriched oil)

A sample of DHA-rich algae oil, equivalent to 250mg, was weighed accurately, placed in a round bottom flask, 10mL of n-heptane was added by using a pipette, and sonicated for 15 minutes with intermittent shaking. 20mL of 0.5N methanolic sodium hydroxide solution was added and connected to the condenser via a Claisen adapter. The contents were refluxed with a magnetic stirrer for 10 minutes and cooled in an ice bath for 5 minutes without removing the round bottom flask. 20mL of boron trifluoride methanol complex solution (13-15%) are slowly and carefully added through a Claisen adapter and refluxed with a magnetic stirrer for another 30 minutes. Cooled in an ice bath for 5 minutes without removing the round bottom flask. 10ml of n-heptane were slowly carefully added via a Claisen adapter and refluxed for a further 5 minutes. The mixture was cooled and the round bottom flask was removed. 5mL of saturated sodium chloride solution was added, shaken and the contents transferred to a centrifuge tube. Centrifuge at low speed (500 RPM) for 5 minutes. The 2mL heptane-free layer was diluted to 50mL with isopropanol and mixed. Separately, 5mL of this solution was diluted to 50mL with methanol and mixed.

The procedure is as follows:

20 μ l of blank solvent (solvent system without analyte), standard solution-1 (containing a known amount of DHA) five replicates, standard solvent-2 (containing DHA samples for analysis (three replicates) and sample solution (single) were injected separately into the chromatograph, the chromatogram recorded and the peak response of DHA measured.

The DHA peak has a retention time of about 11.5 minutes.

Applicability of the system:

1) For the main peak, the column efficiency should not be lower than 2000 theoretical plates.

2) The theoretical plate of the main peak should not exceed 2.0.

3) The relative standard deviation of the area response of the main peak in five replicate injections of the standard solution should not exceed 2.0%.

4) The asymmetry (tailing factor) under each condition does not exceed 2.0.

Table 11: validation of the development of HPLC method for analysis of DHA

Claims

1. An oral omega-3 fatty acid enriched oil emulsion composition for use in the management of a treatment condition to overcome said condition.

2. The oral omega-3 fatty acid rich oil emulsion composition according to claim 1, wherein the omega-3 fatty acid rich oil is a microalgae oil comprising 40% DHA and the emulsion comprises DHA in the range of 50-100mg/ml.

3. The oral omega-3 fatty acid rich oil emulsion composition according to claim 1, wherein all components are natural and biocompatible components suitable for administration to children including newborns.

4. The oral omega-3 fatty acid rich oil emulsion composition of claim 1, wherein the size of the dispersed phase is in the nanometer range.

5. The oral omega-3 fatty acid rich oil emulsion composition of claim 1, wherein it has stability at room temperature (about 30 ℃) and refrigeration temperature (2-8 ℃).

6. The oral omega-3 fatty acid rich oil emulsion composition of claim 1, wherein said emulsion comprises therapeutically effective amounts of vitamins and minerals.

7. The oral omega-3 fatty acid rich oil emulsion composition of claim 1, wherein said disorder is selected from the group consisting of: premature birth disorders in pregnant women, cognitive disorders in children and cardiovascular disorders.

8. The oral omega-3 fatty acid rich oil emulsion composition of claim 7, wherein:

a. the premature birth hurdle of pregnant women is overcome by achieving normal delivery,

b. cognitive impairment in children is overcome by improving their cognitive abilities, an

c. Cardiovascular disorders are overcome by restoring a healthy state.

9. An oral thixotropic emulsion of omega-3 fatty acid rich oil having improved absorption in the nanometer size range, comprising an emulsion in the nanometer size range having improved surface area and absorption according to claim 4.

10. The oral omega-3 fatty acid rich thixotropic emulsion of claim 9, comprising natural emulsifiers and derivatives thereof and biosurfactants, alone or in combination: vitamins, minerals, natural ingredients Generally Regarded As Safe (GRAS).

11. The oral omega-3 fatty acid rich thixotropic emulsion of claim 10, wherein the natural emulsifying agent comprises one or more selected from the group consisting of: natural gums, clays, and polymers.

12. The oral omega-3 fatty acid rich thixotropic emulsion of claim 11, wherein the composition comprises an additive further comprising one or more selected from the group consisting of: rheology modifiers, antioxidants, preservatives, stabilizers, sweeteners and flavouring agents.

13. An oral omega-3 fatty acid rich oil emulsion composition having a nano-sized dispersed phase comprising a natural emulsifier and derivative thereof surfactant, alone or in combination with: vitamins, minerals, natural ingredients Generally Regarded As Safe (GRAS).

14. The oral omega-3 fatty acid-rich oil emulsion of claim 13, wherein the natural emulsifier comprises a natural gum.

15. The oral omega-3 fatty acid rich oil of claim 13, wherein said composition comprises an additive further comprising one or more selected from the group consisting of: rheology modifiers, antioxidants, preservatives, stabilizers, sweeteners and flavouring agents.

16. A High Performance Liquid Chromatography (HPLC) method for the determination of omega-3 fatty acids.

17. A High Performance Liquid Chromatography (HPLC) method for the determination of omega-3 fatty acids according to claim 11, said method comprising the steps of:

a. injecting blank, standard solution-1, standard solution-2 and sample solution into chromatograph,

b. recording a chromatogram and

c. the peak response of docosahexaenoic acid (DHA) was measured.

18. The High Performance Liquid Chromatography (HPLC) method for the determination of omega-3 fatty acids of claim 17, wherein: (ii) (a) blank injected is a single repeat, (b) standard solution injected-1 is five repeats, (C) standard solution injected-2 is two repeats, and (d) sample solution injected is a single repeat, (e) column used is Thermo Syncronis C18 (250x4.6 mm), 5 μm or equivalent, (f) pump mode is isocratic, (g) flow rate is 1.0ml/min, (h) detection at UV 210nm, (i) injection volume is 20 μ l, (j) column oven temperature is 45 ℃, (k) run time is 20 min.

19. A High Performance Liquid Chromatography (HPLC) method for the determination of omega-3 fatty acids according to claim 18, wherein:

a. the solution-1 comprises a DHA working standard,

b. the solution-2 comprises a DHA test solution,

c. sample solutions included a known amount of algae oil rich in DHA sonicated with n-heptane in a round bottom flask for a period of time, added methanolic sodium hydroxide solution and refluxed with a stirrer for 10 minutes, cooled in an ice bath without removing the round bottom flask, slowly and carefully added boron trifluoride methanol complex solution, further refluxed with a magnetic stirrer, cooled in an ice bath without removing the round bottom flask, carefully and slowly added n-heptane and refluxed, cooled the mixture and removed the round bottom flask, added saturated sodium chloride solution, shaken up and the contents transferred to a centrifuge tube, centrifuged at low speed, diluted with isopropanol and the heptane layer was mixed, further diluted with methanol and mixed.

20. The oral omega-3 fatty acid rich oil emulsion according to any one of claims 1, 9 and 13, wherein:

a. the natural emulsifier includes one or more selected from the group consisting of

i. Vitamin E TPGS (d- α -tocopheryl polyethylene glycol 1000 succinate);

phospholipids comprising one or more selected from the group consisting of soy phosphatidylcholine and egg phosphatidylcholine, distearoylphosphatidylcholine, phosphatidylethanolamine and phosphatidylserine;

gums including one or more selected from the group consisting of gum arabic, guar gum, xanthan gum, and gum tragacanth, and

a polymer comprising one or more selected from the group consisting of pectin, gelatin and algin,

b. an emulsion stabilizer comprising one or more selected from the group consisting of xanthan gum, guar gum, gum arabic, bentonite, glycerin, and mixtures thereof,

c. the oral omega-3 fatty acid rich oil comprises one or more selected from the group consisting of microalgae oil, fish oil or linseed oil,

d. an antioxidant comprising one or more selected from the group consisting of butylated hydroxytoluene, rosemary oil, sodium ascorbate, vitablend (consisting of vitamin E and ascorbyl palmitate), sodium metabisulfite, ascorbyl palmitate and vitamin E,

e. vitamins selected from the group consisting of oil soluble vitamin A, vitamin D, vitamin E, and vitamin K; one or more of the group consisting of water-soluble vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, folic acid and vitamin C; minerals including zinc, copper, magnesium, potassium, calcium such as calcium phosphate or calcium carbonate, iron, beta-carotene, and the like,

f. a buffer comprising one or more selected from the group consisting of sodium carbonate citrate and phosphate buffers; a flavoring agent comprising one or more flavorings selected from the group consisting of orange, strawberry, raspberry, mango, peach, vanilla, lime flavorings,

g. a sweetener comprising one or more selected from the group consisting of sorbitol, xylitol, mannitol, sucralose, stevia, aspartame, neotame, acesulfame potassium and mixtures thereof

h. Preservatives, including rosemary extract, sodium benzoate, sodium azide, methyl paraben and propyl paraben.

21. A process for the preparation of an oral omega-3 fatty acid rich oil emulsion comprising the steps of:

a. preparing an oil phase of omega-3 fatty acid rich microalgae oil by mixing a natural emulsifier, a vitamin blend comprising an antioxidant and a DHA rich microalgae oil in a manufacturing tank with a stirrer at room temperature

b. Preparing the aqueous phase in a tank with a stirrer, comprising the steps of:

i. soaking the colloid in purified water for a period of time required for dissolution,

dissolving vitamin E TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate) in another container under mechanical agitation and mixing other water soluble ingredients including preservatives and high intensity sweeteners with this solution,

thereafter, mixing the gum and both the vitamin E and ascorbyl palmitate solutions under mechanical agitation for a period of time required to form a homogeneous mixture,

thereafter, the oil phase is added to the aqueous phase under mechanical stirring, the two phases are maintained at room temperature,

v. adding a flavoring agent, and

continue stirring for an additional period of time.

22. The oral omega-3 fatty acid rich oil emulsion of any one of claim 1, claim 9, claim 13 and claim 21, wherein:

a. the natural emulsifier comprises one or more selected from the group consisting of

i. Vitamin E TPGS (d- α -tocopheryl polyethylene glycol 1000 succinate);

a phospholipid selected from one or more of the group consisting of soy and egg phosphatidylcholine, distearoyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine;

gums consisting of gum arabic, guar gum, xanthan gum, and tragacanth gum, and

a polymer consisting of pectin, gelatin and algin,

b. an emulsion stabilizer comprising one or more selected from the group consisting of xanthan gum, guar gum, gum arabic, bentonite, glycerin and mixtures thereof,

c. an antioxidant comprising one or more selected from the group consisting of butylated hydroxytoluene, rosemary oil, sodium ascorbate, vitablend (consisting of vitamin E and ascorbyl palmitate), sodium metabisulfite, ascorbyl palmitate and vitamin E,

d. vitamins selected from the group consisting of oil soluble vitamin A, vitamin D, vitamin E, and vitamin K; one or more of the group consisting of water-soluble vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, folic acid and vitamin C; minerals including zinc, copper, magnesium, potassium, calcium such as calcium phosphate or calcium carbonate, iron, beta-carotene, and the like,

e. a buffering agent comprising one or more selected from the group consisting of sodium citrate and sodium carbonate,

f. a flavoring agent comprising one or more flavorings selected from the group consisting of orange, strawberry, raspberry, mango, peach, vanilla, lime flavorings,

g. a sweetener comprising one or more selected from the group consisting of sorbitol, xylitol, mannitol, sucralose, stevia, aspartame, neotame, acesulfame potassium and mixtures thereof,

h. a preservative comprising one or more selected from the group consisting of rosemary extract, sodium benzoate, sodium azide, methyl paraben and propyl paraben.

23. Process for the preparation of an oral omega-3 fatty acid rich oil emulsion according to claim 17,

a. the natural emulsifier comprises a soybean phosphatidylcholine compound and a natural emulsifier,

b. the oil phase comprises the following mixture:

i. omega-3 fatty acid-rich microalgae oil 12.5-25% w/v,

antioxidant mixture (Vitablend) ^TM ) 0.05 to 0.5 percent of,

butylated hydroxytoluene 0.1%,

12.5-25% w/v of DHA-enriched microalgae oil,

v. the manufacturing tank is stainless steel jacketed,

vi stirring at 40-50 ℃ at a speed of 100-300RPM,

said gum is 0.3-1.5% w/v xanthan gum),

soaking in purified water at 40-50 ℃ for a period of 1-5h,

1-5% w/v vitamin E TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate), 1000-1500RPM mechanical agitation, preservative sodium benzoate 0.02-0.1% w/v,

x. the high intensity sweetener is sucralose 0.1-0.5%,

homogenizing the mixture by stirring and mixing both the gum and vitamin E TPGS solution at 40-50 ℃ with mechanical agitation at 1000-1500RPM for 30-60 minutes,

finally, the oil phase is added to the aqueous phase with mechanical stirring at 1000-1500rpm, the two phases are kept at 40-50 ℃,

xiii. The added seasoning is

0.5 to 1.0% w/v orange oil, and stirring for 1-2 hours.